Project abstract Current available treatments for Alzheimer's disease (AD) only provide modest amelioration of cognitive and behavioral decline. Recent clinical trials targeting amyloid-? (A?) production or clearance did not show efficacy prompting a reexamination of approaches to AD treatment. Brains from AD patients have been shown to exhibit accumulation of ceramide, a signaling molecule and an integral component of exosomal membranes. One major source of ceramide is through the hydrolysis of sphingomyelin catalyzed by neutral sphingomyelinase 2 (nSmase2). Even though transient increases in ceramide through nSMase2 upregulation are part of normal brain functioning, experimental evidence indicates that chronic nSMase2 upregulation results in negative effects including neuroinflammation and oxidative stress. Recent studies also implicate nSMase2 in both A? aggregation and tau protein propagation through exosome secretion from neurons and glial cells. Moreover, inhibition of exosome synthesis by genetic or pharmacological inhibition of nSMAse2 significantly reduced A? aggregation and tau propagation both in vitro and in vivo thus opening a new avenue for AD therapeutics. While nSmase2 is emerging as an important player in AD etiology, the current armamentarium of nSMase2 inhibitors is inadequate to develop potential treatments. Currently available inhibitors have limitations including low potency (IC50's in M level), poor solubility, and limited brain penetration. In order to address these limitations, we developed a human nSMase2 high throughput screening assay and screened over 350,000 compounds which led to the identification of several hits belonging to different chemical series. Early optimization of two of these hits from different chemical series led to two potent compounds with IC50s of 50 and 300 nM. Both of these compounds were confirmed as inhibitors of exosome release and exhibited good pharmacokinetic profiles and brain penetration (AUCbrain/AUCplasma = 0.27 and 0.6). The objective of this proposal is to further optimize these nSMase2 inhibitors to identify a potent, selective, brain penetrable candidate to carry out proof of concept before future IND enablement studies and ultimately for treatment of patients with AD. Aim 1 is to conduct structure-activity relationship studies to improve potency. Aim 2 is to characterize the compounds from aim 1 for functional inhibition of exosome release, metabolic stability, selectivity and in vivo pharmacokinetics. Aim 3 is to carry out in vivo proof of concept studies by evaluating selected compounds in the 3xTg mouse model of AD. The work proposed involves a novel therapeutic target which is mechanistically distinct from previous efforts in AD treatment, has the potential of addressing disease progression, and exploits two newly discovered chemical series of drug-like nSMase2 inhibitors.